Pregnancy is associated with dramatic increases in uterine blood flows correlating with fetal growth and survival. The uteroplacental unit has prominent production of estrogen, NO and eNOS expression. Estrogen increases NO production. Uterine Artery Endothelial Cells (UAEC) of Pregnancy (P) show specific "Programmed Adaptation" [not seen in Nonpregnant (NP)-UAEC] that maintains in cell culture passages. "Programmed Adaptation" thus is a UAEC response of freshly isolated UA Endothelium ex vivo, that upon many passages does not diminish. The overall hypothesis of this proposal is that: In pregnancy, shear stress/flow in the presence of estrogen causes "Programmed Adaptation" of the UAEC to modulate important endothelial functions associated with NO-mediated vasodilator production. We will give specific emphasis to those mechanisms that increase eNOS activation, eNOS expression, UAEC signaling kinases (e.g. ERK 1/2),and cell-cell communication (measured as synchronized Ca2+ bursts) as modulated by VEGF and Gapjunctions. Aim 1: Effects of prolonged Laminar Shear Stress with and without E2(3on expression of key functional proteins (eNOS, COX-1, PGIS, cPLA2, CX43, ERa, ERp, VEGFR-1, VEGFR-2, and NP-1) necessary for adaptation in UAECs from Luteal Phase Nonpregnant Sheep (NP-UAEC) and Late Pregnant Sheep (P- UAEC). Aim 2: Effects of prolonged Laminar Shear Stress with and without E2pon "Programmed Adaptation" in UAECs from NP-UAEC and P-UAEC. Three "Programmed Adaptation Specific Markers" include: a)ATP/VEGF- stimulated eNOS activation; b) ATP/VEGF-stimulated ERK1/2 phosphorylation; and c) ERK-MAPK and Gap junction mediated ATP-induced Ca2+ bursts. Aim 3: Effects of Estrogen Receptor antagonism with ICI 182,780 on the expression of key functional proteins (as in Aim 1) necessary for Shear Stress/E2p adaptation of UAECs. Aim 4: Effects of Estrogen Receptor antagonism with ICI 182,780 on Shear Stress/E2p UAEC "Programmed Adaptation Specific Markers" (as in Aim 2). Aim 5: Evaluate the effect of prolonged Laminar Shear Stress on development of programmed endothelial cell functions comparing responses of endothelial cells derived from human Embryonic Stem Cells (Project III) to human Embryonic Stem Cells (Project IV; Core B) themselves. These studies will provide a mechanistic framework for understanding interactions between shear stress and estrogen to regulate NO production via VEGF and gap junction mediated cell-cell communication. Vascular adaptations in pregnancy are important because increases in fetoplacental/uteroplacental perfusion, are linked directly to fetal growth and these mechanisms are dysfunctionalin pathologic pregnancies (e.g. preeclampsia/ IUGR).